For most infections, the immune system confers long-term and sometimes life-long protection against many microbial pathogens. One encounter with a microbial pathogen is sufficient to allow the immune system to remember this pathogen for decades to come. This fundamental property forms the basis for vaccination with the main purpose of eliciting long-term protective immunity. Several good vaccines exist, but they were derived quite empirically and we still do not understand the molecular mechanisms that make them so successful. Vaccine preparations based on live attenuated pathogens are notoriously more effective than killed preparations. Our increasing knowledge of the critical elements involved in the initiation of immune responses points us towards two central players, antigen presenting cells (APCs) and pattern recognition receptors (PRRs). APCs capture microbial pathogens and mobilize a sequence of events that forms the basis for mounting an effective immune response against the pathogen. Adjuvants, used in most vaccine preparations, are a mixture of microbial components that trigger PRR activation and signal the transcriptional initiation of immune response genes. Our studies so far provide strong support for differential innate immune recognition of live and dead bacteria. While live bacteria trigger production of inflammatory alert cytokines, dead bacteria fail to do so. Thus, a component differentially present in live and dead bacteria, and which correlates with microbial viability, appears to be uniquely recognized by APCs. Recognition of this component requires the TLR signaling adaptor MyD88. We hypothesize that the innate immune system is capable of discriminating between live and dead pathogens, and accordingly mobilizes distinct signaling pathways and cellular immune responses. The unique responses triggered by live bacteria may hold the key to long-term immunity. Our specific aims are designed to: 1) Determine the ability of live bacteria to activate cytosolic sensors of infection. 2) Characterize the nature of intracellular compartments carrying live versus dead bacteria. PUBLIC HEALTH RELEVANCE: This proposal is aimed to understand the basis for a new mode of immune recognition, the innate sensing of microbial viability. We aim to identify those pathways specifically triggered by live and not dead pathogens. Our work will help set the stage for novel generations of vaccines that hold the promise of conferring protection against many new and existing microbial threats.